1. Field of the Invention
The present invention relates in general to a method and devices for rapid cooling of small (10 microliter or smaller) biological samples to cryogenic temperatures (approximately 150 K or less). The method and devices minimize the thickness of the cold gas layer that forms above the surfaces of liquids and solids (e.g., copper) cooled to cryogenic temperatures, replacing this cold gas layer with gas at another, warmer and more uniform temperature at the same or similar pressure.
2. Description of the Background Art
Cryopreservation of proteins, cells, tissues and other biological samples plays an important role in modern biology and medicine. For example, proteins extracted from natural sources or obtained from genetically engineered organisms and dissolved in an aqueous (water-containing) buffer are often frozen in liquid nitrogen at its boiling or vaporization temperature Tv=77 K and then stored in liquid nitrogen, in dry ice (Tv=195 K) or cryogenic freezers (T≈195 K) to prevent them from degrading. However, the freeze-thaw cycle often causes changes in protein structure that affect protein function, as well as protein aggregation and precipitation.
Cryopreservation of sperm is essential for propagation of animals by artificial insemination, in human fertility treatments, and in preservation of endangered species. Current methods typically involve an initial slow cooling over dry ice (Tv=195 K) followed by more rapid cooling in liquid nitrogen to T=77 K. Sperm survival rates and especially fertilization rates after cryopreservation and thawing are highly variable and often extremely poor.
Cryopreservation is also crucial to protein crystallography, by which the molecular structure of proteins is determined. Protein and virus crystals are easily damaged by the X-rays used to measure their structures. This damage is greatly reduced by cooling the crystals to T=120 K or below. Diffusion of hydroxyl radicals, hydrogen radicals and other reactive species created by X-ray absorption is then limited, reducing their ability to attack the protein. Since these crystals contain large amounts of water, the frozen water provides a rigid framework that limits molecular motions in response to damage. Current methods used for cryoprotection involve soaking the crystals in cryoprotective agents and then cooling by inserting the crystal into a cold gas stream or plunging it into liquid nitrogen or propane. These methods damage the crystals, reducing the accuracy and detail of the molecular structure that can be obtained from X-ray diffraction measurements on them.
Common cooling agents used in cryopreservation to remove heat energy from samples and maintain them at low temperatures include dry ice (Tv=195 K); closed cycle cryogenic refrigerators (T≈195 K); cold gas streams at T˜100 K (nitrogen) or T˜20 K (helium); liquid nitrogen at its boiling point (Tv=77 K); hydrocarbons such as propane and ethane at temperatures just above their melting points (Tm=90 K and 83 K, respectively); and cryogenic refrigerants (chlorofluorocarbons (CFCs) and their modern replacements) that remain liquid below T=200 K. Liquids generally provide more efficient heat transfer and cooling than gases. Liquids having a large difference between their melting temperature and boiling temperature (such as propane (Tv=184 K, ΔT=94 K) and ethane (Tv=231 K, ΔT=148 K) and held just above their melting temperature can absorb more heat from a sample before vaporizing, than, e.g., nitrogen (Tm=63 K, ΔT=14 K). Vapor evolved around a warm sample when it is plunged into the liquid insulates the sample from the liquid, reducing cooling rates; liquids like propane and ethane reduce the amount of vapor evolved and generally give larger heat transfer rates. For liquid samples, the gas evolution problem can be eliminated by freezing on a cold metal surface, but cooling on metal surfaces is less effective and/or damaging for protein crystals, cells and tissues.
Protein solutions, protein crystals, cells and tissues all contain large amounts of water, and so both the final sample temperature and the cooling rate to that temperature are important in determining the properties of the frozen sample and the success of cryopreservation. If water is cooled slowly, it will form crystalline (usually hexagonal) ice. The growth of ice crystals as the water freezes may puncture cell walls, rupture protein crystal lattices, and cause other damage to biological samples. If water is cooled rapidly below its glass transition temperature Tg≈136 K, crystalline ice formation can be avoided and the water will instead form an amorphous, vitreous or glassy state.
Pure water can only be vitrified with cooling rates approaching 106 K/s, which are only achievable for samples with very small volumes (<10−6 microliters) and with very large surface area-to-volume ratios. Cryoprotective agents (CPAs) like glycerol, ethylene glycol, dimethyl sulfoxide (DMSO), polyethelene glycols (PEGs), sugars and even proteins (at very high concentrations) inhibit crystalline ice formation and allow the vitreous phase to be obtained at higher temperatures and using smaller cooling rates. For sufficiently high CPA concentrations (e.g., 60% glycerol), the vitreous ice phase can be obtained even with very slow cooling, and at temperatures accessible using dry ice or −80° C. (193 K) refrigerators. Cryoprotective agents are thus widely used in cryopreservation of biological samples.
However, cryorprotective agents, especially at large concentrations, can cause osmotic shock to the sample leading to cell rupture or crystal cracking, changes in protein conformation, and other chemical and physical changes that degrade the sample before cooling and/or during subsequent warming. Thus, higher sample cooling rates are desirable to reduce the cryoprotective agent concentrations required to obtain vitreous ice.
Higher cooling rates are also desirable to maintain sample integrity and homogeneity during cooling, so as to capture and preserve the sample's initial native structure and function observed at, e.g., room or body temperature. Essentially all physical and chemical properties of the sample—including protein conformation and solubility, salt solubility, pH, and the activity of water—vary with temperature. Changes in these properties that occur during the time the sample is cooling can lead to precipitation of salt or protein, conformational heterogeneity of proteins, changes in membrane structure, and other problems so that the structure of the cryopreserved sample deviates from the initial structure. By cooling very rapidly, little time is allowed for the sample's constituents to respond to the changing temperature before all motion is frozen out. Rapid cooling may thus more accurately preserve the sample's native structure.
In protein cryocrystallography, crystals are cooled by insertion into a cold nitrogen gas stream at T≈100 K or by plunging into liquid nitrogen at its boiling point or liquid propane just above its melting point. Reported cooling rates are ˜300-1500 K/s, so that the time for the crystal to cool from 273 K to 120 K is of order 0.1 to 1 s. Even when substantial concentrations of cryoprotectants (e.g., 30% glycerol) are used, these modest cooling rates result in substantial crystal damage as evident from X-ray diffraction measurements. This damage is a major factor limiting the quality of the resulting molecular structure. In at least some cases, the frozen protein structure differs in important ways from the room-temperature, biologically relevant structure. These modest cooling rates achieved in protein crystallography are still extremely large compared with those in standard protocols for cryopreservation of protein solutions, cells (e.g., sperm) and tissues, which are typically in the range of 0.1 to 10 K/s.
The fastest reported cooling rates of biological samples have been achieved in the field of cryoelectron microscopy. By plunging ˜0.1 micron thick samples (produced by microtoming) supported on thin metal grids into liquid ethane at high speeds (˜5 m/s), cooling rates up to roughly 300,000 K/s can be achieved. The Vitrobot, sold by FEI in Germany, is the most advanced commercial device for freezing samples for cryoelectron microscopy. Protein crystals, cells and tissues all have much larger dimensions and much smaller surface-to-volume ratios than the ultra-thin electron-transmissive samples used in cryoelectron microscopy. They cannot survive such high speed impacts with liquid cryogens, and the splashing when they (and their sample holders) impact the liquid cryogen is problematic.
The fastest cooling of water-containing liquid samples has been achieved either by shooting very small (10−5 microliter) drops into vacuum, which then cool by evaporation, or by spraying small drops in vacuum onto cold metal surfaces. These methods can both be used to vitrify pure water, without added cryoprotective agents. The need for sending samples through vacuum (or low pressures) complicates cooling apparatus and protocols, and the evaporation and resulting dehydration of samples required for evaporative cooling may not accurately preserve the native sample structure. Evaporative cooling in vacuum is more effective than cooling in liquids or on solids only for very small samples (10−5 microliter). Consequently, exposure of larger samples to vacuum or reduced pressures prior to or during a plunge into a cold liquid can cause slower cooling and more cooling related damage than when samples move through gas at atmospheric pressure (whose high thermal conductance and thermal mass minimizes sample cooling even the presence of evaporation.)
The inventors have investigated cooling by immersion (plunging) in liquid nitrogen (at Tv=77 K) and liquid propane (at T just above Tm=90 K) of drops of mixtures of water and common cryoprotective agents as a function of the drop volume. Drops with smaller volumes have larger surface-to-volume ratios and are thus expected to cool at higher rates. Smaller drops should then require smaller concentrations of cryoprotective agents in order for them to cool into the vitreous ice phase.
For a given CPA and for sample plunge speeds into the liquid of roughly 0.5 m/s, the minimum required cryoprotectant (e.g., glycerol) concentration was found to decrease with decreasing sample volume, as expected, for volumes above 0.1 microliters. But contrary to expectations, as the drop volume was decreased below 0.1 microliters, the minimum required cryoprotectant concentration remained roughly constant and large (roughly 28% w/v for glycerol). When drops were placed on an ultra-thin copper foil cup and then the bottom of the cup was plunged into contact with liquid nitrogen, the cryoprotectant concentration to achieve vitrification decreased monotonically with drop volume down to the smallest volumes examined (10−4 microliters). This indicated that the saturation in cryoprotective agent concentration observed when drops were directly plunged into the cryogen was due to a saturation of the cooling rate with volume below 0.1 microliters (at that plunge speed). Since protein crystals used in molecular structure determinations by X-ray crystallography have volumes of 10−2 to 10−7 microliters, this saturation in cooling rate in conventional plunge cooling explained why smaller crystals have not, until now, shown significantly less damage on cooling, significantly better diffraction quality or required significantly less cryoprotectants than larger crystals.